Project Summary: Invariant natural killer T (iNKT) cells are innate-like ?? T cells that use conserved T cell receptor (TCR) rearrangements to recognize lipid antigens in the context of the major histocompatibility complex (MHC) I-like molecule CD1d. Analogous to the classical Th1, Th2, and Th17 helper T cell subsets, NKT1, NKT2, and NKT17 cells have been described with stereotypical cytokine production profiles, transcription factor expression, and tissue localization. The vast majority of iNKT cells in C57BL/6 mice are NKT1 cells, which reside in the liver and spleen and produce high amounts of IFN? and IL-4 when activated by a combination of TCR and innate cytokine signals. Many reports have described iNKT cells in the context of proinflammatory immune responses where they respond to danger signals and microbial lipid antigens to mediate host defense. In contrast, we recently identified a distinct role of iNKT cells in adipose tissues of mice and humans where they display a unique regulatory phenotype. Maintenance of the non-inflammatory state in adipose tissue is essential to preserve insulin sensitivity, prevent diabetes, and control obesity. We found that adipose iNKT cells produce high levels of IL-2 that drives the expansion of adipose Tregs and IL-10 that drives M2 macrophage expansion. These iNKT cell functions contribute significantly to the anti-inflammatory adipose microenvironment, and in the absence of iNKT cells mice are prone to obesity and diabetes. Furthermore, specific activation of adipose iNKT cells with the lipid antigen ?-galactosylceramide (?GalCer) induces weight loss and ameliorates many metabolic abnormalities caused by obesity. Interestingly, adipose iNKT cells have a distinct transcriptional profile and lack expression of the PLZF transcription factor, which is expressed in iNKT cells in all other organs. Instead, they express the transcription factor E4BP4 that drives their production of IL-10. We have several lines of evidence to indicate that the regulatory properties of adipose iNKT cells are induced by exposure to the adipose microenvironment. The goal of this proposal is to identify components of adipose tissue that drive the unique regulatory phenotype of adipose iNKT cells. In line with this goal, we propose to first characterize the ability of adipose tissue to induce and maintain the regulatory iNKT cell phenotype in vivo using adoptive transfer experiments (Aim 1). Then, we will analyze the contribution of adipocyte CD1d expression to the phenotype of adipose iNKT cells (Aim 2). Finally, we will identify molecules released by adipose tissue induce E4BP4 expression in iNKT cells and endow them with regulatory capacity (Aim 3). Combined, these studies will identify environmental and molecular inducers of regulatory iNKT cells that will 1) reveal new pathways to target these cells in autoimmune diseases and 2) offer insight into the biology of an adipose-resident immune cell population that can be manipulated for the treatment of metabolic diseases such as obesity and diabetes.